www.nature.com/scientificreports OPEN Diferential mechanisms of action of the trace amines octopamine, synephrine and tyramine on the Received: 15 March 2019 Accepted: 29 June 2019 porcine coronary and mesenteric Published: xx xx xxxx artery Andy Hsien Wei Koh 1, Russ Chess-Williams1,2 & Anna Elizabeth Lohning1 Trace amines such as p-tyramine, p-octopamine and p-synephrine are found in low concentrations in animals and plants. Consumption of pre-workout supplements containing these plant-derived amines has been associated with cardiovascular side efects. The aim of this study was to determine the mechanisms of action of these trace amines on porcine isolated coronary and mesenteric arteries. Noradrenaline caused contraction of mesenteric arteries and relaxation of coronary arteries. In both tissues, all three trace amines induced contractions with similar potencies and responses were unafected by the β-adrenoceptor antagonist propranolol (1 µM), the nitric oxide synthase inhibitor L-NNA (100 µM), or the TAAR-1 antagonist, EPPTB (100 nM). However, the contractile responses of mesenteric arteries, but not coronary arteries, were signifcantly reduced by depletion of endogenous noradrenaline. Mesenteric responses to all three amines were abolished in the presence of prazosin (1 µM) whereas residual contractile responses remained in the coronary artery which were inhibited by a high concentration (100 µM) of EPPTB. The results suggest complex responses of the coronary artery to the trace amines, with activity at α1-adrenoceptors and potentially TAARs other than TAAR-1. In contrast the actions of the amines on the mesenteric artery appeared to involve indirect sympathomimetic actions and direct actions on α1-adrenoceptors. p-Tyramine (tyramine), p-octopamine (octopamine) and p-synephrine (synephrine) are substituted phenethyl- amines with a phenolic hydroxyl group in the para-position (Fig. 1). Tey are found in nanomolar concentrations in the mammalian nervous system and have thus been described as “trace amines”1. Tey also occur naturally in citrus plants such as Citrus aurantium2. Since 2004, Citrus aurantium extracts have been marketed as ergogenic and weight-loss aids, but there are limited studies to support this claim3. Te safety of Citrus aurantium-listed supplements is still debated as adverse cardiovascular efects have been associated with their use4. A number of actions of these trace amines on cardiovascular tissues has been reported. In some vascular tissues, tyramine has been shown to act as an indirectly acting sympathomimetic agent, promoting the release of endogenous noradrenaline5. Moreover, tyramine and octopamine cause nitric oxide dependant vasodilatory responses in pre-contracted rat mesenteric vascular beds6. Synephrine and octopamine are weak direct agonist 7–9 efects at the α1-adrenoceptors of the isolated rat aorta . In addition, octopamine and synephrine have been shown to exert weak direct β1-adrenoceptor agonist efects on isolated cardiac tissues and to activate cloned 10,11 β2-adrenoceptors . Tus, the amines can potentially exert cardiovascular efects via both direct or indirect mechanisms, but the extent of these actions in diferent vessels of the same species has not been explored. Recently it has been recognised that these amines may exhibit some activity mediated via trace amine-associated receptors (TAARs)12,13. Six subtypes of this receptor have been identifed, but knowledge of their distribution and functions is limited. All three amines have been shown to activate human TAAR-1 expressed in cloned cells and increase cAMP levels, with tyramine being the most potent drug12–14. It has been hypothesised 1Faculty of Health Sciences and Medicine, Bond University, 4229, Queensland, Australia. 2Centre for Urology Research, Faculty of Health Sciences and Medicine, Bond University, 4229, Queensland, Australia. Correspondence and requests for materials should be addressed to A.E.L. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:10925 | https://doi.org/10.1038/s41598-019-46627-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports OH H NH2 N HO HO Tyramine Synephrine OH OH NH2 HO NH2 HO HO Octopamine Noradrenaline Figure 1. Chemical structures of the trace amines (tyramine, octopamine, and synephrine) and noradrenaline. Figure 2. Initial cumulative-dose responses of trace amines and noradrenaline on isolated mesenteric artery. Te amines shown are tyramine (● n = 6), synephrine (■, n = 6) and octopamine (▲, n = 6). Te data are presented as percent of contraction to 60 mM potassium chloride (%KCl). Welch corrected unpaired t-test, *, p < 0.05 vs. tyramine; ^, p < 0.05 vs. synephrine. that tyramine and octopamine acts directly on TAARs to cause vasoconstriction in endothelium-denuded rat aortas15,16 and pig coronary arteries17, but no selective antagonists were available to investigate this hypothesis. Te presence of specifc tyramine receptors had previously been proposed to explain rat aortic responses to syn- ephrine that were resistant to conventional receptor antagonists8. Tus, there is some indirect evidence to suggest that tyramine and synephrine may induce some vascular responses via specifc receptors. Te aim of the present study was to investigate the actions and mechanisms of action of the trace amines on the mesenteric and coronary artery using a porcine model. Te mesenteric artery is densely innervated by the sympathetic nerves, and is a major regulator of blood fow to the gastrointestinal system during physiological stress18. Blood fow to the intestine can be drastically reduced (>80%) during exercise which results in the shunt- ing of blood to the skeletal and cardiac muscle19. In contrast, during physiological stress the adrenergic nervous system ensures an increased blood fow to cardiac muscle via the coronary circulation20,21. Tus, in this study, the relative potencies of the trace amines tyramine, octopamine and synephrine were investigated on the vascular tone of mesenteric artery and the coronary artery and the mechanisms of action for each amine in the two func- tionally very diferent arteries determined. Te role of TAAR-1 in responses was also examined using EPPTB (RO-5212773), a recently developed selective antagonist for TAAR-122. Results Responses of the porcine mesenteric artery. All three trace amines produced concentration-depend- ent contractions of the porcine arterial rings (Fig. 2). Tyramine and synephrine produced similar maximum responses, whilst those to octopamine were signifcantly greater (unpaired Welch’s t-test, p < 0.05). All three trace amines had similar potencies that were not signifcantly diferent (pEC50 ranging from 3.25–3.91). Te endog- enous amine, noradrenaline produced greater contractions than the three trace amines and was also the most potent drug tested (Fig. 2, Table 1). Responses of the porcine coronary artery. All three trace amines produced concentration-dependent contractions of porcine coronary arterial rings (Fig. 3). Octopamine and synephrine produced similar maximum responses, whilst those to tyramine were greater, the diference between tyramine and octopamine being statis- tically signifcant (p < 0.05). Te potencies of all three amines were similar and ranged from 3.30–3.88 (Table 2). SCIENTIFIC REPORTS | (2019) 9:10925 | https://doi.org/10.1038/s41598-019-46627-5 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Controls Treatment Trace amine and interventions Max contraction (%KCl) Potency (pEC50) Max contraction (%KCl) Potency (pEC50) Sample number (n) Tyramine Tyramine pre-treatment (3 mM) 30.0 ± 3.9 3.91 ± 0.14 2.9 ± 0.7* 2.87 ± 0.06* 6 Propranolol (1 µM) 2.9 ± 0.7 2.49 ± 0.19 2.9 ± 0.6 2.88 ± 0.27 6 Prazosin (1 µM) 2.9 ± 0.7 2.49 ± 0.19 Abolished* Abolished* 6 L-NNA (100 µM) 4.2 ± 2.7 2.50 ± 0.23 6.5 ± 3.0 2.81 ± 0.56 4 Synephrine Tyramine pre-treatment (3 mM) 25.6 ± 4.8 3.81 ± 0.12 10.9 ± 2.6* 3.76 ± 0.26 6 Propranolol (1 µM) 12.5 ± 2.9 3.90 ± 0.51 16.1 ± 3.8 3.45 ± 0.41 6 Prazosin (1 µM) 9.6 ± 2.6 3.81 ± 0.56 Abolished* Abolished* 5 L-NNA (100 µM) 19.2 ± 10.1 4.06 ± 0.68 17.4 ± 9.8 2.29 ± 1.19 4 Octopamine Tyramine pre-treatment (3 mM) 63.4 ± 6.3 3.25 ± 0.12 19.9 ± 2.3* 3.60 ± 0.21 5 Propranolol (1 µM) 17.1 ± 3.6 3.80 ± 0.44 15.4 ± 4.4 3.46 ± 0.52 5 Prazosin (1 µM) 14.0 ± 2.8 3.80 ± 0.45 Abolished* Abolished* 6 L-NNA (100 µM) 16.7 ± 9.3 3.80 ± 0.44 12.8 ± 6.0 3.55 ± 0.41 6 Noradrenaline Tyramine pre-treatment (3 mM) 85.7 ± 12.2 5.42 ± 0.21 85.9 ± 6.5 5.22 ± 0.13 5 Propranolol (1 µM) 81.9 ± 5.9 5.92 ± 0.14 91.4 ± 4.4 5.65 ± 0.09 5 Prazosin (1 µM) 87.7 ± 4.7 5.92 ± 0.14 81.9 ± 5.2 5.10 ± 0.08* 5 Table 1. Efects of antagonists on the maximum responses and potency values of p-synephrine, octopamine, tyramine, noradrenaline and phenylephrine in porcine mesenteric arteries. Paired Student’s t-test *p < 0.05 vs control. Te endogenous amine noradrenaline failed to produce contraction and only relaxations were observed (Fig. 3). Tese relaxations were converted to contractions in the presence of the β-adrenoceptor antagonist propranolol (1 µM). Te maximum contractile responses to noradrenaline (33.4 ± 2.7% of the response to potassium) were signifcantly greater than those to octopamine (20.3 ± 4.6%, p < 0.05) and synephrine (21.1 ± 7.3%, p < 0.05), but not tyramine (32.3 ± 4.5%). In the presence of both propranolol (1 µM) and the α1-adrenoceptor antagonist prazosin (1 µM) responses to noradrenaline were abolished completely. The role of endogenous noradrenaline release in responses in mesenteric artery. In mes- enteric arteries, tyramine pre-treatment for 30 minutes nearly abolished subsequent responses to tyramine with a > 90% reduction in maximal contraction and a rightward shif of curves (pEC50 value 2.87 ± 0.06, n = 6; Student’s t-test p < 0.05) (Fig.